Abstract

Purpose: Corneal inflammation associated
with ocular adenoviral infection is caused by leukocytic infiltration
of the subepithelial stroma in response to expression of interleukin-8
(IL-8) and monocyte chemoattractant protein-1 (MCP-1) by infected
corneal cells. We have shown that these two chemokines are activated by
the mitogen-activated protein kinases (MAPKs) extracellular
signal-regulated kinase (ERK) and p38 for IL-8, and Jun-terminal kinase
(JNK) for MCP-1. It is also well established that transcription of each
of these chemokines is tightly controlled by the nuclear factor kappa B
(NFκB) transcription factor family. Therefore, we sought to better
understand the differential regulation of chemokine expression by NFκB
in adenoviral infection of the cornea.

Methods: Primary keratocytes derived
from human donor corneas were treated with signaling inhibitors and
small interfering RNA specific to MAPKs, and infected with adenovirus
for different time periods before analysis. Activation of specific NFκB
subunits was analyzed by western blot, confocal microscopy,
electromobility shift assay, and chromatin immunoprecipitation, and
chemokine expression was quantified by enzyme-linked immunosorbent
assay.

Results: Upon adenoviral infection, NFκB
p65, p50, and cREL subunits translocate to the nucleus. This
translocation is blocked by inhibitors of specific MAPK signaling
pathways. Confocal microscopy showed that inhibitors of the p38, JNK,
and ERK pathways differentially inhibited NFκB nuclear translocation,
while PP2, an inhibitor of Src family kinases, completely inhibited
NFκB nuclear translocation. Western blot analysis revealed that
activation of specific NFκB subunits was time dependent following
infection. Chromatin immunoprecipitation experiments indicated that
binding of NFκB p65 and p50 subunits to the IL-8 promoter upon viral
infection was differentially reduced by chemical inhibitors of MAPKs.
Electromobility shift assay and luciferase assay analysis revealed that
transactivation of IL-8 occurred with binding by the NFκB p65 homodimer
or NFκB p65/p50 heterodimer as early as 1 h post infection, whereas
MCP-1 expression was dependent upon the NFκB cREL but not the p65
subunit, and occurred 4 h after IL-8 induction. Finally, knockdown of
NFκB p65 by short interfering RNA abrogated IL-8 but not MCP-1
expression after adenoviral infection.

Conclusion: The kinetics of NFκB subunit
activation are partly responsible for the observed pattern of acute
inflammation in the adenoviral-infected cornea. MAPKs differentially
regulate chemokine expression in adenoviral keratitis by differential
and time-dependent activation of specific NFκB subunits.

An acute inflammatory response to infection or injury occurs in
stereotyped stages irrespective of invading organism or mechanism of
injury, with neutrophils being the first cells to infiltrate the tissue
or body cavity, followed shortly by monocytes [1]. This pattern
appears to be the result of the specific induction and activity of
chemokines, proteins elicited by cells that induce the directed
migration of leukocytes into tissue sites of inflammation [2], by infected or
injured cells. Possible molecular mechanisms at play in the tightly
controlled pattern of acute inflammation include transcriptional
induction, transcriptional repression, and mRNA stability. In
particular, it has been shown that AU-rich elements in mRNA contribute
stability to the molecule and in part serve to control the kinetics of
gene expression of proinflammatory cytokines [3]. Leukocyte
infiltration into the corneal stroma represents a critical pathogenic
event in viral infection of the cornea. Interleukin-8 (IL-8) is one of
the earliest chemokines to be expressed in infection and acts as a
first line of defense via its capacity to elicit neutrophil chemotaxis,
and to a lesser degree monocyte and T-cell chemotaxis [4-6]. IL-8 induction
following viral infection has been shown by many independent research
groups [7-10], and a wide
variety of cells produce IL-8, including microglia and astrocytes [11-13]. However, in the
corneal stroma the molecular mechanisms that regulate IL-8 expression
following adenoviral infection remain unclear. Our study focuses on the
kinetics of transcription of IL-8 and monocyte chemoattractant protein
1 (MCP-1), another key chemokine in adenoviral keratitis, and on the
role of the NFκB transcription factor family in chemokine activation.

The nuclear factor-κB (NFκB) family of transcription factors
controls expression of well over one hundred genes, the majority of
which participate in regulating innate and adaptive immunity [14,15]. Activation of
NFκB occurs within minutes after an appropriate stimulus and leads to
strong transcriptional stimulation of both viral and cellular genes [7,16-18]. Analysis of the
transcriptional regulation of chemokines induced by viral infection is
critical to understanding the pathogenesis of viral keratitis. However,
the mechanisms that connect viral infection to chemokine expression by
infected stromal cells are poorly understood [7,19-22].

In general, chemokine gene expression is controlled by the NFκB
transcription factor family, p65, RELB, cREL, NFκB1 (p50/100), and
NFκB2 (p52/105). These proteins form specific homo- or heterodimers for
transcriptional activation of target genes in a cell-specific manner.
NFκB subunit activation can be achieved through two main pathways:
canonical (classical), characterized by the activation of the IκB
kinase (IKK) complex, including both IKKα and IKKβ; and non-canonical
(non-classical), characterized by activation of NFκB-inducing kinase
and IKKα, but not IKKβ [23-28]. Therefore, it is
the specific activation of upstream IKKs that represents the point of
divergence for NFκB activation. Activation of these pathways has been
determined to be both cell and stimulus specific [26-28]. The canonical
pathway is the route most commonly activated by pathogens, and is
stimulated by pathogen-associated molecular patterns and cytokines. The
non-canonical pathway has been described particularly in B lymphocytes,
and is stimulated by B-cell activating factor, lymphotoxin β, and CD40L
[27,29].
Lipopolysaccharide from Salmonella enterica was shown to
activate both canonical and non-canonical pathways in primary B cells
with activation of both NFκB p50/RELA and p52/RELB heterodimers [26]. Herpes simplex
virus type 1 ICP27 protein was shown to activate NFκB via the canonical
pathway [30].
While the role of NFκB in apoptosis following adenoviral infection has
been explored [31],
its role in cytokine regulation due to viral infection has been less
fully addressed.

We have earlier shown that NFκB p65 is activated upon adenoviral
infection in conjunction with the phosphoinositide 3-kinase/protein
kinase B (PI3K/AKT) pathway for cell survival during viral replication [31], but activation of
NFκB p65 as a possible mechanism for chemokine induction in adenoviral
infection has not been explored. Our prior studies in a mouse model of
adenoviral keratitis have shown expression of KC (a homologue of IL-8
in the mouse) within 4 h of infection, followed by MCP-1 at 16 h post
infection [32].
In this report we address the role of specific NFκB subunit activation
in the kinetics of IL-8 and MCP-1 expression in adenoviral-infected
human corneal cells.

Cell culture and viruses

Primary keratocytes were derived from donor corneas as previously
described [33].
Briefly, after mechanical debridement of the corneal epithelium and
endothelium, corneas were cut into 2 mm diameter sections, and each
section placed in individual wells of 6-well Falcon Tissue Culture
Plates (Fisher Scientific, Pittsburgh, PA) with Dulbecco’s Modified
Eagle Medium (DMEM), containing 10% heat inactivated fetal bovine serum
(FBS), penicillin G sodium, and streptomycin sulfate. Corneal fragments
were removed prior to monolayer confluence. Cells were grown and
maintained at 37 oC in 5 % CO2. Cells from
multiple donors were pooled, and the cell monolayers used at passage
three. For inhibitor analysis, cells were pretreated with SB203580 (10
µM), SP600125 (25 µM), PD98059 (25 µM), and PP2 (10 µM) at 37 °C for 3
h before infection, and were exposed to the inhibitors at the same
concentrations throughout the infection process. The protocol for use
of corneas from deceased human donors was approved by the Massachusetts
Eye and Ear Infirmary Human Studies Committee, and conformed to the
tenets of the Declaration of Helsinki. Human adenovirus species D type
19 (HAdV-D19) used in this study was cultured directly from the cornea
of a patient with EKC [33],
and purified by cesium chloride gradient. The virus was grown in human
lung carcinoma cells (A549 cells, CCL 185; American Type Culture
Collection, Rockville, MD) in Minimum Essential Media (MEM) with 2 %
FBS, penicillin G sodium, streptomycin sulfate, and amphotericin B. The
State of Oklahoma Department of Health confirmed the viral serotype.
Typical adenoviral cytopathic effect, positive immunofluorescent
staining for adenovirus hexon proteins, and increasing titers of virus
within one week after infection of human corneal cells were seen (data
not shown).

Viral infection

Monolayer cell cultures were grown to 95% confluence, serum starved
overnight, inhibitor treated for 3 h in Opti-MEM (Invitrogen, Carlsbad,
CA), and infected at a multiplicity of infection of 50 or mock infected
with virus-free dialysis buffer as a control.

Transfection

Transient transfections were done in 6 well plates using FuGENE 6
(Roche, Indianapolis, IN) as per the manufacturer’s instructions. A
total of 2 μg DNA was transfected, including NFκB p65 or control siRNA
(Imgenex, San Diego, CA), IL-8 luciferase construct, and pRL-TK
construct, the latter to measure renilla luciferase activity as an
internal control. The transfection mixture was prepared by mixing 3 μl
of FuGENE 6 in 47 μl of serum-free DMEM, incubated at room temperature
for 5 min, the DNA added, and further incubated for 15 min, prior to
transfection of 70–80% confluent cells. Viral infections were carried
out 48 h post-transfection, and supernatants and cell lysates collected
at various time points after infection for enzyme-linked immunosorbent
assay (ELISA) and luciferase assay, respectively.

Enzyme-linked immunosorbent assay (ELISA)

The cell supernatants were collected at various time points up to 4
h post infection, and the levels of IL-8 and MCP-1 quantified by
sandwich ELISA. The detection limit was 30 pg/ml. Plates were read on a
SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, CA) and
analyzed with SOFTmax analysis software (Molecular Devices). The means
of triplicate ELISA values for each of the viral- and mock-infected
wells were compared by one-way ANOVA with preplanned contrasts, with
α=0.05.

Electrophoretic mobility gel shift assay (EMSA)

Nuclear extracts from adenoviral- or mock-infected keratocytes were
prepared using Nucbuster (Novagen, Madison, WI), and binding and
supershift assays done using the LightShift Chemiluminescent EMSA kit
(Pierce), according to the manufacturers’ instructions. Briefly,
chemokine sense and antisense oligonucleotides encoding specific
binding sites for NFκB were synthesized (IDT, Coralville, IA).
Oligonucleotides were then labeled using Biotin-ddUTP and terminal
transferase for 15 min at 37 °C in the labeling buffer and
then annealed. For the assay, 10 μg of nuclear extract, labeled
oligonucleotide, poly (dI-dC; 1 μg), and poly L-lysine (0.1 μg) were
mixed in the binding buffer and incubated at room temperature for 15
min. For comparison, 100 molar excess of unlabelled probe was added to
the reactions 15 min before the addition of labeled probe. For the
supershift assay, 1 or 2 μg antibody to NFκB p65, p50, or cREL was
added to the binding reaction and incubated on ice for 30 min.
Protein-DNA complexes were resolved in 5% pre-electrophoresed
polyacrylamide gel in 0.5× TBE running buffer and then transferred to a
nylon membrane (Roche, Indianapolis, IN). The membrane was then probed
for anti-biotin and the bands were detected by chemiluminescence using
Kodak films and developed in a QCP X-Ray film processor in which each
film is exposed for a preset time. Densitometric analysis of EMSA was
performed using ImageQuant 5.2 (Pierce, Rockford, IL) in the linear
range of detection, and absolute values then normalized to binding in
mock-infected cells. The means of normalized replicate EMSA values for
each condition were compared by one-way ANOVA with preplanned
contrasts, with α=0.05.

Confocal microscopy

Keratocytes grown on chamber slides (Nunc, Rochester, NY) were
treated with dimethyl sulfoxide or inhibitor for 3 h and then infected
with adenovirus or dialysis buffer for 20 min. Cells were partially
fixed in 0.05% paraformaldehyde for 10 min, washed in PBS containing 2%
FBS, and permeabilized in solution containing 0.1% Triton X-100 for 5
min. After 30 min blocking in 3% FBS-PBS, the cells were incubated in 5
μg/ml of NFκB p65, p50, and cREL primary antibody for 1 h at room
temperature, washed, and incubated in Alexafluor-594 and Alexafluor-488
conjugated secondary antibody (Molecular Probes, Eugene, OR) for 1 h
more at room temperature. Cells were then washed, fixed in 2%
paraformaldehyde, and mounted using Vectashield (Vector labs,
Burlingame, CA) mounting medium containing DAPI. Images were taken with
an Olympus FluoView 500 confocal microscope using a 60× water immersion
objective.

Activation of NFκB subunits and their inhibitory kinase (IκB) in
adenoviral infection

In the absence of stimulation, NFκB components are sequestered in
the cytoplasm by a tight association with inhibitory proteins of the
IκB family. Upon stimulation, IκB is phosphorylated by IKK-containing
complexes, releasing NFκB subunits and leading ultimately to the
degradation of IκB via the ubiquitin-proteosome pathway [27], and translocation
of NFκB to the nucleus for specific transcriptional activity. We
infected human keratocytes for 1 and 4 h and performed immunoblot
analysis for phosphorylation of specific NFκB subunits, IKKα/β, and
IκB. After 1 h, adenoviral infection induced phosphorylation of NFκB
p65 and p50, IKKα/β, and IκB but not cREL (Figure 1A). Phosphorylation of
p65, IKKα/β, and IκB was reduced by inhibitors of p38 MAPK (SB203580),
ERK (PD), and Src (PP2; Figure 1A). However, the JNK
inhibitor (SP600125) only partially reduced the activation of NFκB p65,
IKKα/β, and IκB, consistent with our previous observation that these
MAPK have different downstream targets in adenoviral-infected cells [7,8,34], possibly because
JNK is not involved in IL-8 induction. At 4 h post infection, we
observed increased cREL phosphorylation in viral-infected cells as
compared to mock-treated cells (Figure 1B). These results indicate
that IκB and members of the NFκB family are activated rapidly upon
adenoviral infection, and that their activation is dependent on
upstream kinases shown previously to be important to chemokine
expression by adenoviral-infected cells [7,8].

In general, NFκB transcription factors are activated rapidly after
exposure to viral infection, resulting in a strong transcriptional
regulation of a multitude of early viral and cellular genes [35]. We previously
reported the activation of NFκB p65 in a PI3K/AKT dependent manner upon
adenoviral infection that ensured delayed cell death and promoted viral
replication [31].
EMSA performed on adenoviral-infected keratocytes at 1 h post infection
showed NFκB binding to the IL-8 promoter as distinguished from mock
infection (Figure
2A). In viral-infected cells, antibody specific to each NFκB
subunit appeared to reduce binding, as indicated by reduced density of
the primary band, demonstrating the possible involvement of NFκB
homodimers or heterodimers in the activation of IL-8 gene expression.
Specificity of probe binding was shown by use of 100 molar excess of
unlabelled probe (data not shown). We have previously shown that
chemical inhibition of JNK has no effect on IL-8 expression by
adenoviral-infected keratocytes. Similarly, the JNK inhibitor appeared
to have no effect on NFκB binding to the IL-8 promoter, while
inhibitors of p38, ERK, and Src reduced NFκB binding (Figure 2A).
Statistically, overall binding was significantly greater in
viral-infected cells as compared to mock-infected cells (Figure 2B,
p<0.0001, ANOVA). IL-8 promoter binding of NFκB subunits was
significantly reduced by all four inhibitors (p<0.05). Antibody
binding/shift on the IL-8 promoter was not observed in mock-treated
cells or in cells treated with any inhibitor prior to infection (Figure 2B,
p>0.05). These data correlated with our western blots (Figure 1)
and our previous observation that JNK regulates expression of MCP-1 but
not IL-8 [34].

At 1 h after infection, we did not observe significant binding of
NFκB subunits to the MCP-1 promoter (Figure 2C). However, at 4 h post
infection, NFκB subunit binding to the MCP-1 promoter was significantly
greater in viral-infected cells as compared to mock-infected cells
(p<0.0001). All 3 subunits, but in particular cREL, bound to the
MCP-1 promoter at 4 h after infection (Figure 2C), indicating that IL-8
and MCP-1 may be regulated at different time points and by different
NFκB dimers. We occasionally but not consistently observed low levels
of NFκB p65 binding to the MCP-1 promoter (data not shown). By
graphical analysis of multiple EMSA experiments, for MCP-1, only the
JNK inhibitor reduced binding, consistent with a JNK-specific
activation pathway for MCP-1 (Figure 2D, p<0.05). For the
MCP-1 promoter, a binding/shift was seen with p50 and cREL (p<0.05),
but not with p65 (p>0.05), suggesting that NFκB p65 does not
participate in MCP-1 transcription. In light of our in vivo data
showing that KC, an IL-8 homolog, is the first chemokine to be induced
upon adenoviral-infection, and that MCP-1 expression is delayed, these
results indicate that in adenoviral infection, IL-8 is transactivated
by NFκB p65/p65 homodimers and/or NFκB p65/p50 heterodimers, while
MCP-1 gene expression is delayed and uses predominantly cREL and NFκB
p50.

Cellular localization of NFκB subunits in adenoviral infection

NFκB and IκB shuttle continually between the cytoplasm and nucleus
in steady state conditions, resulting in a basal level of NFκB activity
[36]. We
previously demonstrated increased nuclear localization of NFκB p65 upon
adenoviral infection [7].
Now, using confocal microscopy, we confirmed increased nuclear
localization of NFκB subunits in viral- but not mock-infected cells (Figure 3A).
MAPK inhibitors partially impaired the nuclear localization of NFκB
p65, p50, and cREL in viral-infected cells (Figure 3A). Interestingly,
SP600125 had less inhibitory effect on nuclear localization compared to
other MAPK inhibitors, indicating that the JNK pathway does not mediate
NFκB p65 activation. The Src inhibitor PP2 completely suppressed NFκB
p65 and p50 nuclear translocation (Figure 3A), suggesting a broad
upstream role of Src in these pathways, while downstream MAPKs
collaborate in the activation and translocation of NFκB. Confocal
microscopy was also performed for the cREL subunit of NFκB. In these
experiments, cREL and p65 were both translocated into the nucleus upon
viral infection (Figure
3B) as compared to mock-treated cells (Figure 3B).
Pretreatment with signaling inhibitors of Src and MAPK reduced both p65
and cREL nuclear translocation, except for SP600125 (Figure 3B).

Specific NFκB subunits bind to the IL-8 and MCP-1 promoters upon
viral infection in a time-dependent fashion

The presence of NFκB in the nucleus is not by itself a direct
indication of transcriptional activity [36]. To confirm whether translocated NFκB p65
is transcriptionally active upon viral infection, we analyzed the
binding of NFκB p65 and p50 to the IL-8 or MCP-1 promoter using a ChIP
technique. Binding of both NFκB p65 and p50 on the IL-8 promoter was
considerably increased with adenoviral infection, well above the basal
levels seen in mock-treated cells at 1 and 4 h post infection (Figure 4A).
As expected, inhibitors of Src and MAPKs dramatically reduced NFκB p65
and p50 binding. Anti-mouse serum control demonstrated the specificity
of NFκB p65 and p50 antibody binding, as the control antibodies did not
pull down IL-8 DNA. IL-8 promoter binding appeared similar at 1 and 4 h
post infection. cREL binding to the IL-8 promoter was negligible.
Interestingly, no NFκB p65 was bound to the MCP-1 promoter at 1 or 4 h
post infection (Figure
4B). NFκB p50 binding to the MCP-1 promoter was equivalent
at both 1 and 4 h after infection and was unaffected by MAPK
inhibitors. A marginal increase in cREL binding to the MCP-1 promoter
was apparent at 1 h post infection, increasing dramatically at 4 h
after infection. cREL binding at 4 h was reduced by all inhibitors
utilized.

NFκ-B p65 is critical for IL-8 induction in adenoviral infection

Given that adenoviral infection induces NFκB p65 translocation,
transcriptional activation, and IL-8 promoter binding, we wished to
determine whether p65 is essential to IL-8 induction in
adenoviral-infected cells. We used NFκB p65-specific or scrambled
control siRNA for these experiments. Figure 5A shows successful
knockdown of NFκB p65 expression in keratocytes to 70– 80% in repeat
experiments (data not shown). When co-transfected with IL-8 luciferase
construct, NFκB p65 specific siRNA reduced IL-8 luciferase activity to
basal levels at all time points tested post infection (Figure 5B,
p<0.0001), while scrambled control siRNA demonstrated no such
effect. Interestingly, MCP-1 ELISA performed on supernatants from the
same experiments (Figure
5C) showed no reduction in MCP-1 protein expression in p65
siRNA treated cells, indicating that NFκB p65 is dispensable for MCP-1
induction. MCP-1 expression became significantly elevated in
viral-infected cells only at 4 h post infection (p<0.0001). siRNA
against p65 did not significantly alter MCP-1 protein expression in
viral-infected cells at any time post infection.

The observed difference in the kinetics of IL-8 and MCP-1 chemokine
expression after adenoviral infection, in the studies herein,
correlates well with the pattern of leukocyte infiltration in vivo [32], but in general,
the mechanisms for differential expression of proinflammatory cytokines
in infection are not well understood. The stepwise order and timely
expression of various inflammatory mediators seem to be preset as a
part of a master gene activation program. For example, the consistent
differential in time between the expression of interferon gamma and
IL-17 during active experimental autoimmune encephalitis in DA rats
suggests different roles for these cytokines in the pathogenesis of the
disease [37]. Our
data is consistent with the idea that the kinetics of cytokine
expression in inflammation are due in large part to the interplay
between elements that regulate transcriptional induction,
transcriptional repression, and perhaps mRNA stability [3].

Two decades after the initial discovery of NFκB, new functions for
this ubiquitous transcription factor family continue to emerge. Recent
progress in understanding how the immune system senses and responds to
pathogens has drawn new attention to NFκB as a key effector of
inflammatory responses to infection [27]. Its multiple actions, redundancy in
function, and cell specificity have made building a picture of the
molecular mechanisms by which NFκB activates cytokine genes in
infection a complex undertaking. Viruses that have been previously
shown to activate NFκB include human immunodeficiency virus [38], hepatitis B virus
[39], hepatitis C
virus [40,41], Epstein Barr
virus [42],
herpes simplex virus [43],
and influenza virus [44].
The K13 protein of human herpes virus-8 was shown to mediate IL-8 via
NFκB p65, p50 and cREL [10].
Most prior studies of the influence of adenoviral infection on NFκB
activation used either recombinant adenoviruses [45], adenovirus
vectors [46,47], or isolated
adenovirus proteins [7,45,48,49]. The possible
roles of NFκB binding kinetics and subunit specificity in chemokine
expression by primary keratocytes infected with a native adenovirus
have not been previously studied.

Many previous reports demonstrate IL-8 induction through various
MAPK pathways converging on NFκB [11,41],
and binding of NFκB p65 to the IL-8 promoter [7,50-53]. In our studies,
adenoviral-infected cells showed increased nuclear translocation and
IL-8 promoter binding for NFκB p65 and p50. Interestingly, we
demonstrated by confocal microscopy, EMSA, and western blot that the
JNK inhibitor (SP600125) had a minimal effect on NFκB p65 and p50
binding to IL-8 promoter. These data correlate with our earlier reports
showing that inhibition of JNK activity in adenoviral-infected cells
did not reduce IL-8 expression [34] or inhibit NFκB p65 translocation to the
nucleus [7].
However, the ChIP data did appear to show an effect of SP600125 on p65.
Also, cREL binding to IL-8 promoter by ChIP assay was negligible, but
our supershift assay with cREL on the IL-8 promoter was significant.
Further studies will be necessary to resolve these apparent
discrepancies.

Our confocal microscopy data demonstrated increased nuclear
translocation of NFκB p65, cREL, and p50 in adenoviral-infected cells,
whereas no such translocation was observed in mock-treated cells. By
confocal microscopy, inhibitors to various MAPKs reduced but did not
completely inhibit NFκB subunit translocation to the nucleus,
suggesting that NFκB nuclear translocation represents a summation of
upstream activity by numerous kinases. Our earlier studies demonstrated
that inhibition of Src fully blocked activation of p38 MAPK, ERK, and
JNK (32, 35, and 52), suggesting that Src kinases are linearly upstream
to all 3 MAPKs. By confocal microscopy, inhibition of Src also
effectively blocked NFκB nuclear translocation.

Although IL-8 is one of the best-studied chemokines in host–pathogen
interactions, the specific signaling and molecular mechanism underlying
the kinetics of its induction remain to be fully elucidated. IL-8
contributes to the chemotaxis of neutrophils but also other leukocytes [11-13], and has become a
paradigm chemokine for translational studies of anti-chemokine therapy.
Monoclonal antibody to IL-8 was recently utilized successfully as a
treatment for localized pustular psoriasis [54]. Our data clearly show that NFκB p65 is
critical for IL-8 expression, as knockdown of p65 reduced IL-8
luciferase activity to mock levels at multiple time points post
infection. Reduced p65 did not inhibit expression of MCP-1, suggesting
again that MCP-1 expression is not NFκB p65 dependent. We suggest that
NFκB p65 and cREL play a role in the kinetics of IL-8 and MCP-1 gene
regulation, respectively, in adenoviral-infected primary keratocytes.
Further studies are needed to clarify the molecular mechanisms
underlying MCP-1 induction and how other transcription factors such as
Sp1 may regulate chemokine induction in adenoviral infection.

Supported in part by U.S. Public Health Service NIH grants EY013124
and P30EY014104, Massachusetts Lions Eye Research Fund, Inc.,
New England Corneal Transplant Research Fund, and an
unrestricted grant to the Department of Ophthalmology, Harvard Medical
School, from Research to Prevent Blindness, New York, NY.